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The basement membrane is important for proper tissue development, stability, and physiology. Major components of the basement membrane include laminins and type IV collagens. The type IV procollagens Col4a1 and Col4a2 form the heterotrimer [a1(IV)] 2 [a2(IV)], which is ubiquitously expressed in basement membranes during early developmental stages. We present the genetic, molecular, and phenotypic characterization of nine Col4a1 and three Col4a2 missense mutations recovered in random mutagenesis experiments in the mouse. Heterozygous carriers express defects in the eye, the brain, kidney function, vascular stability, and viability. Homozygotes do not survive beyond the second trimester. Ten mutations result in amino acid substitutions at nine conserved Gly sites within the collagenous domain, one mutation is in the carboxy-terminal noncollagenous domain, and one mutation is in the signal peptide sequence and is predicted to disrupt the signal peptide cleavage site. Patients with COL4A2 mutations have still not been identified. We suggest that the spontaneous intraorbital hemorrhages observed in the mouse are a clinically relevant phenotype with a relatively high predictive value to identify carriers of COL4A1 or COL4A2 mutations.

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Epigenetic perturbations are assumed to be responsible for phenotypic abnormalities of fetuses and offspring originating from in vitro embryo techniques. We studied 29 viable Day-80 bovine fetuses to assess the effects of two in vitro fertilization protocols (IVF1 and IVF2) on fetal phenotype and genomic cytosine methylation levels in liver, skeletal muscle, and brain. The IVF1 protocol employed 0.01 U/ml of FSH and LH in oocyte maturation medium and 5% estrous cow serum (ECS) in embryo culture medium, whereas the IVF2 protocol employed 0.2 U/ml of FSH and no LH for oocyte maturation and 10% ECS for embryo culture. Comparisons with in vivo-fertilized controls (n=14) indicated an apparently normal phenotype for IVF1 fetuses (n=5), but IVF2 fetuses (n=10) were significantly heavier (19.9%) and longer (4.7%), with increased heart (25.2%) and liver (27.9%) weights, and thus displayed an overgrowth phenotype. A clinicochemical screen of 18 plasma parameters revealed significantly increased levels of insulin-like growth factor 1 (40.8%) and creatinine (37.5%) in IVF2, but not in IVF1, fetuses. Quantification of genomic 5-methylcytosine (5mC) by capillary electrophoresis indicated that both IVF1 and IVF2 fetuses differed from controls. We observed significant DNA hypomethylation in liver and muscle of IVF1 fetuses (-16.1% and -9.3%, respectively) and significant hypermethylation in liver of IVF2 fetuses (+11.2%). The 5mC level of cerebral DNA was not affected by IVF protocol. Our data indicate that bovine IVF procedures can affect fetal genomic 5mC levels in a protocol- and tissue-specific manner and show that hepatic hypermethylation is associated with fetal overgrowth and its correlated endocrine changes.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.The Dickkopf family of secreted proteins consists of four members, which share two conserved cysteine-rich domains (12,24). The hallmark of Dkk proteins is that they function as Wnt antagonists or agonists by binding to and inhibiting or activating the Wnt coreceptor LRP6 (1, 31, 45). They show regionalized expression during vertebrate embryogenesis (5,10,13,18,20,33,46). Dkk1 is the best-characterized member of the family. It acts as an embryonic head inducer, and when overexpressed it will induce extra heads in Xenopus and zebra fish (6,12,18,22,36,46). dkk1 mutant mice are embryonic lethal, and embryos lack anterior head structure and display fused digits (36). dkk2 mouse mutants are viable but show bone defects (28). Little is known about the biological role of dkk4.By a number of criteria, dkk3 appears as a divergent member of the dkk family. (i) By DNA sequence similarity, vertebrate dkk1, -2, and -4 are more related to each other than they are to dkk3 (12). (ii) Hydra has two dkk genes, one related to vertebrate dkk1, -2, and -4 (16) and one related to vertebrate dkk3 (9). This suggests an ancient phylogenetic separation between these family members, where dkk1, -2, and -4 but not dkk3 arose by gene duplication from an ancestral dkk (16). (iii) Soggy is a protein of unknown function with sequence similarity to dkk3 but not to other dkk genes (24). The similarity is most pronounced outside the two conserved Dkk cysteine-rich domains, raising the possibility that the gene arose from an ancestral dkk3 precursor. (iv) Unlike Dkk1, -2, and -4, Dkk3 does not act as a Wnt modulator (24,29,55). While all other tested Dkk proteins bind to and modulate the Wnt receptor LRP6, as well as the Dkk coreceptor Kremen, Dkk3 has no affinity to these transmembrane proteins (7,30,32,33), and no other proteins are known to interact with it.Like other dkk members, dkk3 is expressed during vertebrate development in suggestive patterns in many organs (7,33). Prominent expression of dkk3 is observed in the brain and in fibroblasts of adult rodents (17,24,34,37,56) and in the human adrenal cortex (50).